Heavy metals occur at various concentrations in different types of soils. In trace amounts certain heavy metals such as copper (Cu) and zinc (Zn) perform vital structural roles as cofactors in enzyme homeostasis. However, when in excess, these heavy metals as well as non-essential metals such as cadmium (Cd), mercury (Hg) and lead (Pb) are toxic. Many human disorders have been attributed to ingestion of heavy metals including an increased rate in cancer in response to Cd.
Remediation of soils containing high levels of heavy metals requires physical removal of the metals, because most of these metals cannot be degraded in the soil, as is the case for certain organic contaminants. Current practical methods used to decontaminate such sites involve physical excavation of top soils, transport and reburial elsewhere. These clean-up methods are only feasible for small areas and are very costly. For example, cleaning one hectare to a depth of one meter can cost between $600,000 and $3,000,000.
Studies have shown that several plant types are effective at taking up significant concentrations of heavy metals from soils and waters (Baker & Brooks, Biorecovery 1: 81-126 (1989) and Dushenkov et al., Environ. Sci. & Tech. 29: 1239-1245 (1995)). These heavy metal "hyperaccumulators" are widespread throughout the plant kingdom. Most metal hyper-accumulating plants have been identified by selecting plants that grow on sites with high metal concentrations (e.g. mining sites). Some of the very efficient heavy metal hyper-accumulators such as Thlaspi caerulescens (Brown, Environ. Soil & Tech. 29: 1581-1858 (1995)) are relatively small or have small root systems and low biomass which limits their ability for removal and storage of large amounts of heavy metals. Brassica juncea plants, which produce larger biomass have been identified as efficient metal hyper-accumulators (Salt et al. Plant Physiol. 109: 1427-1433 (1995).
Initial studies indicate that removal of heavy metals from soils by plants would be orders of magnitude less costly and could become efficient by maximizing metal uptake through biological engineering (Cunningham & Ow et al., Plant Physiol. 110: 715-719 (1996); Brown et al., J. Env. Qual. 23: 1151-1157 (1994); and Salt et al., Plant Phys. 109: 1427-1433 (1995)). Several rate-limiting steps are critical for effective removal of heavy metals from soils. These include making the contaminants biologically accessible in the soil (by chelation and acidification) and subsequent uptake of heavy metals across the plasma membrane of root cells. Furthermore, upon entering plant cells, intracellular detoxification is achieved by production of appropriate high-affinity ligands or chelation proteins and peptides. Several mechanisms of intracellular detoxification/chelation have been identified in plants including metallothionins (Hamer, Annu. Rev. Biochem. 55: 913-951 (1986) and Maitani, et al. Plant Physiol. 110: 1145-1150 (1996)), glutathione-derived phytochelatins (Howden et al. Plant Physiol. 107: 1059-1066 (1995) and Grill et al. Science 230: 674-676 (1985)), or metal binding amino acids (Kraemer et al. Nature 379: 635-638 (1996)).
A recent study has shown that reduction of the heavy metal Hg to its non-charged metallic form, which is less toxic, significantly reduces plant toxicity and can enhance removal of Hg (Rugh et al. Proc. Nail. Acad. Sci. USA 93: 3182-3187 (1996)). In a following detoxification step, heavy metal-peptide complexes are shuttled into the plant lysosomal vacuolar organelles (Ortiz et al. EMBO J. 11: 3491-3499 (1992) and Ortiz et al. J. Biol. Chem. 270: 4721-4728 (1995)). An ATP binding cassette (ABC)-type transporter has been shown to mediate vacuolar sequestration of heavy metals in yeast. Sequestration of conjugated heavy metal-peptide complexes in the large plant vacuoles effectively removes these compounds from various metal-sensitive enzymes in the plant cell cytoplasm (Salt et al. Plant Phys. 107: 1293-1301 (1995)). In addition, it is considered advantageous if heavy metals are further transported into shoots and leaves of plants before being sequestered into vacuoles as these aerial parts of the plant are more amenable to harvesting for heavy metal removal (Cunningham et al. Plant Physiol. 110: 715-719 (1996)). Identification of transporters that load heavy metals into the vascular tissue in roots and that enable heavy metal uptake into leaf cells from the vascular system will be essential for biological engineering of root to shoot transfer.
Plant plasma membrane cation uptake transporters have been isolated by complementation of yeast mutants deficient in accumulation of specific cationic nutrients (Sentenac et al. Science 256: 663-665 (1992); Anderson et al. Proc. Natl. Acad. Sci. USA 89: 3736-3740 (1992); Ninnemann et al. EMBO J. 13: 3464-3471 (1994); and Schachtman and Schroeder, Nature 370: 655-658 (1994)). Complementation of K.sup.+ uptake deficient yeast mutants led to isolation of first K.sup.+ channel cDNAs in plants, named AKT1 and KAT1 (Sentenac et al. and Anderson et al. supra). In voltage clamp experiments these two cDNAs were shown to encode inward-rectifying K.sup.+ channels that provide a pathway for proton-driven low-affinity K.sup.+ uptake (Schachtman, et al. Science 258: 1654-1658 (1992); Hoshi, The Journal of General Physiology 105: 309-328 (1995); Bertl, Folia Microbiologica 39: 507-509 (1994)).
Despite these advances, genes encoding heavy metal transporters in the plasma membranes of plant cells have not yet been isolated. Identification of genes encoding transporters that load heavy metals into the vascular tissue in roots and that enable heavy metal uptake into leaf cells from the vascular system will be essential for biological engineering of root to shoot transfer. The present invention addresses these and other needs.